Acanthamoeba keratitis is a painful and debilitating disease of the cornea of the eye caused by an amoeba that is ubiquitous in nature. The disease, which is associated with contact lens use, only has been widely recognized since the mid-1980's. Medical treatment is problematical and surgical intervention often is required. Seven species of Acanthamoeba have been implicated as causative agents. Stromal and epithelial forms of the disease have been recognized, but it is unknown whether the form of the disease is related to the amoebic species involved. Classification of these organisms at the species level is a major problem that needs to be solved; a more reliable method of classification may have important implications for the prevention and treatment of the disease. This project proposes using variations in nucleotide sequences of small ribosomal subunit RNA genes (srDNA) to classify Acanthamoeba isolates. Sequences will be determined for a representative sample of clinical isolates following PCR amplification of nuclear srDNA. Phylogenetic information will be used to group strains into clusters with related sequence (ribosets). The sequence and phylogenetic information then will be used to design genus- and species-specific oligonucleotide diagnostic probes. Genus-specific probes will be used in initial screens with dot blots to identify rDNA that has been amplified from samples by PCR or with in situ hybridization to localize and identify amoebas in tissues. Both approaches are very sensitive and should be able to detect single amoebas. Species identification primarily will be based on restriction fragment length polymorphisms (fingerprints) obtained from PCR-amplified rDNA subfragments.